Optical microscopy involves passing light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. The typical system of lenses and imaging equipment, along with the appropriate illumination equipment, sample stage and support, make up the optical microscope. Typical standard optical microscopy, bright field microscopy, suffers from limitations which include that it can only image dark or strongly refracting objects effectively, diffraction limits resolution to approximately 0.2 μm in the visible region, and out of focus light from points outside the focal plane reduce image clarity.
Optical microscopy of biological specimens, particularly live cells, is difficult as they generally lack sufficient contrast to be studied successfully; typically the internal structures of the cell are colourless and transparent. Commonly, contrast is increased by staining the different structures with selective dyes, but this involves killing and fixing the sample. Staining may also introduce artifacts; apparent structural details caused by the processing of the specimen and are thus not a legitimate feature of the specimen.
Within the prior art these limitations have been overcome to some extent by specific microscopy techniques that can non-invasively increase the contrast of the image. In general, these techniques make use of differences in the refractive index of cell structures. These include:                Oblique illumination—wherein side illumination gives the image a 3-dimensional appearance and can highlight otherwise invisible features;        Dark field—wherein directly transmitted light entering the image plane is minimized thereby collecting only the light scattered by the sample;        Dispersion staining—wherein an optical technique results in a colored image of a colorless object, where five different microscope configurations are used which include brightfield Becke line, oblique, darkfield, phase contrast and objective stop dispersion staining;        Phase contrast—where differences in refractive index appear as differences in contrast within the image;        Differential interference contrast—also known as Nomarski contrast microscopy wherein differences in optical density appear as differences in relief an exploits polarization differences near refractive index boundaries;        Interference reflection microscopy—used to examine the adhesion of cells to a glass surface, using polarized light of a narrow range of wavelengths to be reflected whenever there is an interface between two substances with different refractive indices;        Fluorescence—wherein certain compounds when illuminated with high energy light emit light of a different lower frequency and is of critical importance since it can be extremely sensitive allowing the detection of single molecules and wherein many different fluorescent dyes can be used to stain different structures or chemical compounds including one particularly powerful method being the combination of antibodies coupled to a fluorophore as in immunostaining;        Confocal—wherein a scanning point of light instead of full sample illumination is used to give slightly higher resolution, and significant improvements in optical sectioning;        Single plane illumination microscopy and light sheet fluorescence microscopy—wherein a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, allows high resolution optical sections to be taken; and        Deconvolution—wherein the point spread function of the microscope imaging system is deconvolved by computer-based techniques in either two-dimensional or three-dimensional domains.        
There are also a multitude of super-resolution microscopy techniques to circumvent the diffraction barrier including for example serial time-encoded amplified microscopy (STEAM). These are typically based upon imaging a sufficiently static sample multiple times and either modifying the excitation light or observing stochastic changes in the image. Additionally the knowledge of and chemical control of fluorophore photophysics are at the core of these techniques by which resolutions of approximately 20 nm are attainable.
Amongst the many biological systems of interest analysed with optical microscopy is the interaction between Actin and myosin, the two key contractile proteins in muscle. Such analyses have been studied for many years in the prior art using different techniques. Amongst such experiments in vitro Motility assays were extensively performed to obtain new information on the molecular mechanism of muscle contraction. Such assays take advantage of the ability to image rhodamine-phalloidin-labeled Actin filaments by fluorescence microscopy as they interact with and are translocated by myosin bound to a coverslip surface. In most studies on single Actin-myosin filament interactions, see for example Sellers in “In vitro Motility Assays with Actin” (Cell Biology Assays: Essential Methods, Ch. 20, Butterworth-Heinemann 2006), Jerry, and Yamada, the two contractile filaments are not imaged simultaneously due to technical challenges. In some studies, however, Actin and myosin filaments were visualized simultaneously by either using fluorescence reagents/labels attached to both Actin and myosin filaments (Yamada) or by using the combination of dark field and fluorescent microscopy techniques, see for example Kalganov et al in “A Technique for Simultaneous Measurement of Force and Overlap between Single Muscle Filaments of Myosin and Actin” (Biochemical and Biophysical Research Communications, Vol. 403, pp 351-356). Imaging both Actin and myosin filaments is important not only for visualization purposes but also for measuring filament overlap during active acto-myosin interactions because it should give new information about cooperative phenomena of myosin cross-bridges in myosin filaments.
Myosin is known as the molecular motor which converts chemical energy into mechanical work. Thus any chemical reagents attached to myosin for imaging purposes may or may not change the ability of myosin to do its work. For this reason it is critical to avoid using fluorescent reagents conjugated with myosin when a study on Actin and myosin interaction is to be done. The inventors in their previous work, see Kalganov Rassier, showed a technique where fluorescent labeling of myosin is not required for simultaneous imaging and force measurement. In that work a standard Nikon immersion dark field condenser was used to create dark field images of myosin filaments. The disadvantage of using the dark field condenser was in necessity to limit substantially the numerical aperture (NA) of the objective to form the dark field image. The objective's low NA caused Actin filaments to appear dark, hardly distinguishable from the background.
Within the prior art Vodyanoy et al in U.S. Pat. No. 7,688,505 entitled “Simultaneous Observation of Darkfield Images and Fluorescence using Filter and Diaphragm” teach to a system employing an annular diaphragm and optical filter which are used for simultaneous observation of darkfield images and fluorescence. The diaphragm provides a variable diameter controlled by a lever and a removable filter which is used to adjust the amount of unfiltered incident light which produces the darkfield images when directed on a sample whilst the removable filter is used to filter light of the particular frequency for producing fluorescence images. However, Vodyanoy does not address the issues identified and discussed supra in respect of the NA of the optical system nor the requirement to use fluorescent reagents.
Accordingly it would be beneficial to provide an imaging technique which would allow simultaneous visualization of single Actin and myosin filaments as well as the filament overlap without requiring fluorescent conjugates for myosin filament visualization. It would be further beneficial for the imaging technique to improve, i.e. increasing, (Actin) filament image brightness contrast and signal-to-noise ratio (SNR).
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.